Whooping Cough, or pertussis, is a highly infectious disease which primarily affects children. In addition to causing respiratory complications, whooping cough may result in nerve damage and high mortality, particularly in children in low socioeconomic groups and in newborn infants without maternal anti-pertussis antibodies. The etiologic agent of pertussis is the gram negative coccobacillus Bordetella pertussis. The bacteria is believed to invade the respiratory tract and induce a toxic state which remains even after the disappearance of the bacteria.
Although world health organizations presently recommend the immunization of infants to prevent the incidence and spread of pertussis, great concern has arisen over the negative effects of various vaccine forms. The toxicity of conventional B. pertussis vaccine formulations causes side effects which vary from simple flushing to permanent neurological damage and/or death. Consequently, reduced use of conventional B. pertussis vaccines has resulted in an increase in the number of pertussis cases.
The most widely used vaccine contains whole, B. pertussis organisms, which are inactivated after treatment at 56.degree. C. for 30 minutes. Since the bacteria are not subjected to any other detoxification treatment, any toxic substance which can withstand the elevated temperature is included in the vaccine and contributes to the occurrence of side effects. Another consequence of this type of vaccine is the formation of a broad spectrum of antibodies as a response upon administration. The sera induced by such vaccines lack high specificity and high protecting potential for use as preventive or therapeutic treatments, and have no value at all as diagnostic materials.
Other pertussis vaccines are prepared from culture supernatant of B. pertussis. However, variabilities in cultivation allow the final composition of the microorganism to vary, and the inactivating agents, glutaraldehyde or formaldehyde, occasionally lead to aggregated materials subject to conversion to active toxic substances upon storage.
Alternate vaccines are prepared from avirulent or toxin deficient Strains. However, these vaccines have proven to be much less protective than those prepared from virulent strains. See Wardlaw et al, J. Med. Micro. Biol., 9:89-100 (1976).
To avoid the side effects caused by whole cell vaccines, research turned to the investigation of the toxic components of the B. pertussis bacteria for use in acellular vaccines. One important component is pertussis toxin (PT), a protein exotoxin which plays a major role in the pathogenesis of whooping cough and is believed to be the major protective antigen of B. Pertussis. [A. A. Weiss et al, Ann. Rev. Microbiol., 40:661 (1986)].
Another component of interest for an acellular B. pertussis vaccine is the antigen, filamentous hemagglutinin (FHA). This antigen, alone or in combination with PT, has been noted to have some protective capacity. See, e.g., U.S. Pat. No. 4,563,303 and European patent application 231,083.
Purification procedures to isolate the PT and FHA antigens from whole cell B. pertussis have been published resulting in varied yields, and purity of components. For example, U.S. Pat. No. 4,563,303 refers to purifying FHA from Bordetella by adsorbing it on a cellulose sulfate, polysaccharide gel bound with dextran sulfate or a cross-linked polysaccharide gel, and eluting FHA therefrom.
J. J. Munoz et al, Inf. Immun., 32:243-250 (1983) and Japanese published application 59.175439A refer to separate fermentation procedures for each of the two factors PT and FHA and a culture medium for production of FHA. The separation and purification methods described in these documents involve laborious and time-consuming extractions, precipitations, centrifugations and dialysis, followed by a chromatographic purification. Such processes demand at least a week for the isolation of a single factor and are clearly unsatisfactory for antigen or vaccine production on a commercial scale.
M. Chazono et al, J. Biol. Stand., 16:83-89 (1988) and European patent application 121,249 refer to PT and FHA, as major substances of the total hemagglutinin fraction excreted by B. pertussis for use in a vaccine formulation in the same ratio as they occur in fermentation broth. For the extraction and purification of the hemagglutinin the fermentation broth was centrifuged to separate off the cell mass, followed by ammonium sulphate mediated precipitation from the supernatant. The precipitate was purified after redissolution via a sucrose density centrifugation, followed by separation and dialysis. The techniques employed in this method involve manipulations of large volumes requiring complex and sophisticated apparatus, such as continuous centrifugation to avoid lengthy exposure to open air and laborious and frequent manipulations. This results in batch to batch variations of the antigenic composition of the product. Finally, the purity of the resulting antigens may still be insufficient due to residual endotoxins and pyrogenicity caused by lengthy exposure to open air.
Y. Sato et al., Infect. Immun., 41:313-320 (1983) describe a purification method employing primarily affinity chromatography. In this system fermentation broth is passed over spheroidal hydroxyapatite at pH of about 8, whereby most of the FHA is adsorbed, while PT is eluted. Centrifugation prior to the filtration over the adsorbent avoids blocking of the adsorbent by cells and debris, which cause poor flow behavior, poor recovery, slow operation and undesirable contamination for the subsequent steps. The eluate containing the PT fraction is then adjusted to a pH of about 6 and passed over another hydroxyapatite column, whereby PT is retained. Both factors are then further treated in fully separate systems. In this procedure both factors, after elution from their hydroxyapatite carrier with an appropriate buffer/salt solution and pooling, are passed over a support modified with an affinity ligand as haptoglobin sepharose or fetuin-sepharose, to which PT is selectively bound while FHA remains unaffected. The purification is then completed by precipitation, and/or another simple chromatographic fractionation and/or dialysis.
R. D. Sekura et al, J. Biol. Chem., 258:14647-14651 (1983) describes an analogous method, restricted to PT alone. In this procedure the extraction from the supernatant obtained by centrifugation of a B. pertussis fermentation broth is accomplished by adding Affi-Gel blue resin in batch to the supernatant at pH 6, separating it from the liquid phase after a contact time of about two days, eluting the PT that has bound specifically to the Cibacron dye (because of its NAD like structure), with an appropriate buffer/salt solution and pooling the active fractions. For the first purification fetuin-agarose is used as affinity support to bind PT selectively; after elution the purification is completed with a simple fractionating chromatography and salting out.
While the chromatographic methods of Sato et al and Sekura et al avoid the very labor intensive and time consuming zonal centrifugation, they still require separation of cells and debris from the fermentation broth. The adsorption from the supernatant in batch, without passing the whole liquid over a chromatographic column as described in Sekura et al, is a practical simplification. However, in that method there is only one target component to be isolated.
Another chromatographic purification of PT alone was reported by M. Svoboda et al, Anal. Biochem., 159:402-411 (1986). PT was adsorbed from the culture supernatant by adding Blue Sepharose (an equivalent to other Cibacron modified supports) in batch for about 12 hours at pH 6.0, filtering the adsorbent and packing it into a chromatographic column. After elution and pooling the PT fraction was immediately loaded on a Phenyl-Sepharose carrier, to which PT, because of its hydrophobic character, and because of the high ionic strength of the medium, was bound. Elution was accomplished with a pH 10 buffer/glycerol mixture at relatively low salt concentration. The pooled fractions were diluted and acidified to pH 5.0, loaded on hydroxyapatite HTP, and separated from remaining PT-fragments by eluting and pooling.
M. Christodoulides et al, Vaccine, :199-207 (1987) refer to the extraction of PT and FHA from B. pertussis culture using dye-ligand chromatography based on the Sekura et al procedure. The pH of the culture fluid was adjusted to 6.0 and Blue Sepharose gel was added. The gel was washed with Tris-HCl, pH 8.0 buffer and bound material eluted with the buffer containing 1.0M NaCl, pH 8.0.
Y. Sato et al, Lancet, pp. 122-126 (Jan. 21, 1984) refer to a pertussis component vaccine prepared by salting out the FHA antigen and LPF-HA antigen in a B. pertussis culture by fractional precipitation, extraction in phosphate; fractionation by ultracentrifugation; pooling of HA fractions; detoxification with formalin; and addition of adjuvant.
Still other purification techniques employ supports for the selective adsorption of PT from a B. pertussis culture supernatant. European Patent Application 140386A refers to the use of denatured human or animal ceruloplasmin as an affinity ligand which may be bound to CNBr activated sepharose, agarose, cellulose or dextran.
The availability of the affinity supports in larger quantities and with a constant and certified quality however remains a major drawback to large scale commercial use of presently available purification methods. This type of support cannot be sterile and therefore always carries the inherent risk of contamination, especially with viruses, unacceptable in production of human medical products.
There remains a continuing need in the art for effective and safe vaccines against whooping cough, as well as convenient diagnostic tools for early detection of B. pertussis infection.